Specific membrane proteins catalyze the transport of classical neurotransmitters into secretory vesicles in preparation for release by regulated exocytosis. The long-term objectives of this proposal are to understand how this transport activity influences transmitter release, information processing and behavior. The program focuses on the vesicular transport of monoamines because these transmitters have a role in major mental illnesses and drug abuse. We have used selection in the neurotoxin MPP+ to isolate a cDNA encoding the vesicular monoamine transporter expressed in the adrenal gland (VMAT1), further implicating this activity in the form of neural degeneration that occurs in Parkinson's disease. The sequences define a novel mammalian gene family that now includes a second vesicular monoamine transporter expressed in the brain (VMAT2) and a vesicular transporter for acetylcholine (rVAChT). This program uses the cloned cDNAs for vesicular neurotransmitter transporters to understand aspects of their function that have relevance for synaptic transmission and human health. The first specific aim is to use an assay that we have recently developed to characterize the role of the transporter in vesicular amine efflux exchange and the action of amphetamines. The second specific aim is to determine the structural basis for substrate recognition, coupling to the driving force for transport delta/mu/H+ and drug interaction using VMAT1/VMAT2 chimeras and site-directed mutagenesis together with flux and drug binding assays. We will also assess whether VMAT2 functions as a monomer. The third specific aim is to determine the role of phosphorylation in the control of VMAT2 activity. The fourth specific aim extends the analysis to the vesicular ACh transporter and addresses issues that may influence its function such as its intracellular localization and posttranslational modification. These studies will both help to understand the molecular mechanism for neurotransmitter packaging and begin to reveal its role in quantal size, drug abuse and neural degeneration.